The present invention relates to recombinant DNA which encodes the BglII restriction endonuclease and modification methylase, and the production of these enzymes from cells containing the recombinant DNA.
Restriction endonucleases are now one of the foremost tools of molecular biology. They occur naturally in a wide variety of bacteria, both eubacteria and archaebacteria (Roberts and Macelis, Nucl. Acids Res. 20: 2167-2180, (1992)), and when purified, can be used to cut DNA in a sequence-specific manner into precise fragments. By use of the large number of restriction enzymes now available commercially, DNA molecules can be uniquely identified and fractionated into component genes.
Restriction endonucleases are a component of restriction-modification (RM) systems, which have been extensively studied for over forty years (Luria and Human, J. Bacteriol. 64: 557-569, (1952); Bertani and Weigle, J. Bacteriol. 65: 113-121, (1953)). The systems usually contain a second component, the modification methylase (Arber and Linn, Ann. Rev. Biochem., 38: 467-500, (1969); Boyer, Ann. Rev. Microbiol. 25: 153-176, (1971)). It is thought that in nature RM systems act as "bacterial immune systems" that destroy foreign DNA entering cells (Smith, PAABS REVISTA 5: 313-318, (1976)). However, individual bacteria have been known to contain as many as seven different RM systems and this apparent redundancy suggests that these systems serve other functions as well (Stein et al., J. Bacteriol. 174: 4899-4906, (1992)).
In the bacterium, the restriction endonuclease scans incoming DNA for a specific recognition sequence and produces double stranded scissions in the DNA molecule (Meselson, Ann. Rev. Biochem. 41: 447-466, (1972)). The modification methylase acts to protect the bacterium's own DNA against the action of its restriction counterpart. The methylase recognizes and binds the same DNA sequence as its corresponding endonuclease; however, instead of cleaving, it methylates a specific residue within the recognition sequence, thereby preventing endonucleolytic binding or cleavage (Smith, Science 205: 455-462, (1979)). In this manner, the bacterial host DNA is rendered completely resistant to cleavage by the restriction endonucleases within the cell.
Initially the term "RM system" was applied only to systems in which the components had been genetically defined. However, the term has now come to refer to any site-specific endonuclease isolated from a bacterium. In many cases the existence of a modification methylase component is assumed without any rigorous proof (Roberts, CRC Crit. Rev. Biochem. 4: 123-164, (1976)).
When it became obvious that a large number of restriction enzymes would be isolated from a variety of bacteria, Smith and Nathans (J. Mol. Biol. 81: 419-423, (1973)) devised a nomenclature that, with minor modifications, is still being used today. A restriction enzyme is named with a three letter designation that abbreviates the genus and species from which it was isolated. When necessary, a fourth letter is added to designate the strain (like Hind). Roman numerals following the system name are assigned to differentiate multiple enzymes from the same source. The prefixes R and M refer to restriction endonuclease or modification methylase, respectively, but are usually not included. When the three letter designation is used without prefix, it is understood to refer to the endonuclease.
Restriction endonucleases, and to a lesser extent, DNA methylases, have become invaluable reagents for genetic engineering. As the field of biotechnology has grown and developed, there has been a growing commercial incentive to mass produce these enzymes. However, mass production of restriction enzymes from their native organisms is often difficult for several reasons. First, many organisms produce several RM systems, and biochemically separating the different products is often problematic. Second, in addition to multiple restriction systems, bacteria can also produce other nucleases and DNA binding proteins which are difficult to remove biochemically from restriction enzyme preparations. Third, the amount of a particular enzyme produced by an organism can be highly variable, and may vary with growth conditions. Finally, some bacteria may be difficult, expensive, or even dangerous to grow. To solve these problems and reproducibly obtain restriction enzymes in abundance, the techniques of genetic engineering have been applied to create highly productive strains of bacteria.
The cloning of RM systems first began in the 1970's and since then those involved with the effort have had to contend with numerous problems. A problem inherent to every cloning project is identifying and isolating the gene(s) of interest. Some RM systems are plasmid borne so it has been relatively easy in these cases to isolate the DNA encoding both the methylase and endonuclease genes and transfer them onto a new vector (EcoRV: Bougueleret et al., Nucl. Acids Res. 12: 3659-3676, (1984); PaeR7: Theriault and Roy, Gene 19: 355-359, (1982); Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80: 402-406, (1983); PvulI: Blumenthal et al., J. Bacteriol. 164: 501-509, (1985)).
The large majority of RM systems, however, are not plasmid encoded. These have been cloned mainly by "shotgun" methods in which the chromosomal DNA is cleaved into small pieces of clonable size using restriction endonucleases or other means. The pieces are then ligated en masse onto a cloning vector and transformed into a suitable bacterial host generating "libraries" in which each gene is represented multiple times. The difficulty is then to find selective procedures to identify those clones carrying the genes of interest out of the thousands that have been generated.
The first selection method successfully used to clone RM systems, inspired by the classic properties of the systems, involved the use of bacteriophage: cells carrying an RM system survived when exposed to phage attack, while those not expressing the RM system did not (HhalI: Mann et al., Gene 3: 97-112, (1978); PstI: Walder et al., Proc. Natl. Acad Sci. USA 78: 1503-1507, (1981)). Unfortunately, this method had limited success. Cloned RM systems are not always expressed in a manner that confers selective survival. In addition, when there are multiple RM systems in a cell, one cannot target the particular RM system one wishes to clone. Furthermore, other cloned functions besides RM systems are able to protect the cells against phage attack and enable them to survive the selection (Mann et al. Ibid; Howard et al., Nucl. Acids Res. 14: 7939-7951, (1986); Slatko et al., Nucl. Acids Res. 15: 9781-9796, (1987)).
A second method, which has had more widespread success, selects for expression of the methylase gene: upon transformation, any plasmid within the library which contains and expresses the cloned methylase gene will methylate its cognate recognition sites. When the library is subsequently selected by cleavage with an endonuclease of the appropriate specificity, the modified plasmids should not be cut and remain viable upon a second transformation step. The other plasmids not containing the methylase gene should be cleaved by the endonuclease and therefore transform at a much-reduced efficiency (Kiss et al., Nucl. Acids Res. 13:6403-6421, (1985); Lunnen et al., Gene 74: 25-32, (1988)). In all RM systems thus far studied, the restriction endonuclease and modification methylase genes lie in close proximity to one another (Wilson, Nucl. Acids Res. 19: 2539-2566, (1991)); therefore, methylase selection often yields a complete RM system. In other cases the selection may yield only the methylase gene; however, it has often been possible to clone a larger or an adjacent fragment containing the endonuclease gene in a second step (see e.g. Kiss et al. Ibid).
In addition to finding a suitable selection procedure, a number of other difficulties have arisen when attempting to clone RM systems. For example, in some systems problems have arisen when the endonuclease gene is introduced into host cells not already protected by modification. If both genes are introduced together on a common DNA fragment the methylase may not adequately protect the host genome from the action of the endonuclease, if, for example, the endonuclease is produced too soon or in too large quantity. Methods have since been devised to clone the two genes separately on different vectors, in order to protect the cell against activity of the endonuclease gene before the latter is introduced (Howard et al. Ibid). The judicious selection of vectors can be essential to the stability of the clone as well as sufficient production of endonuclease (Brooks et al., Nucl. Acids Res. 19: 841-850, (1991)).
Yet another obstacle to cloning RM systems in E. coli manifested itself during the attempt to clone and express diverse methylases. E. coli has a number of systems that restrict foreign DNA containing heterologous cytosine and/or adenine methylation (Raleigh and Wilson, Proc. Natl. Acad. Sci. USA 83: 9070-9074, (1986); Noyer-Weidner et al., Mol. Gen. Genet. 205: 469-475, (1986); Waite-Rees et al., J. Bacteriol. 173: 5207-5219, (1991)). These systems became apparent only during attempts to clone a variety of methylase genes and establish them in genetically marked E. coli strains. The problem has been overcome in some cases by generating E. coli mutants deficient in these systems. Problems still persist in cloning RM systems; however, as more systems are studied, better cloning methods are being developed.
BglII, from the Gram-positive bacterium Bacillus globigii, was one of the first RM systems to be identified and characterized. (Wilson and Young, in Schlessinger (ed.), Microbiology-1976, American Society for Microbiology, p. 305-357, (1976)). As the name implies, there exists another RM system, BglI, isolated from the same species Both BglI and BglII systems behave in a classic manner, protecting the bacterium against infection by unmodified bacteriophages. Investigators found it very difficult to separate R.BglI from R.BglII by biochemical means since the enzymes have similar molecular weights and ionic properties (Pirrotta, Nucl. Acids Res. 3: 1747-1760, (1976)); therefore, genetic methods were employed. B. globigii was mutagenized by standard methods, and individual isolates were screened for reduced levels of phage restriction using unmodified phage propagated on Bacillus subtilis, a closely related strain. Two mutant strains, designated B. globigii RUB561 and RUB562, producing only R.BglI or R.BglII, respectively, were generated in this manner (Duncan et al., J. Bacteriol. 134: 338-344, (1978)). The mutant strains still maintained methylase functions for both BglI and BglII. The investigators thought the use of mutagenesis coupled with screening for phage restriction would be a generally applicable method for the separation and purification of RM system components. However, since then, the discovery of RM systems has far outpaced the development of genetic systems in diverse bacteria and the method has not been applied to other RM systems. Nonetheless, the two B. globigii strains have been used commercially to produce BglI and BglII endonucleases. In addition, both strains have proved essential in the cloning of the BglII RM system, as described below.